Detailed Workflow
LAMP
Loop-mediated Isothermal Amplification
Loop-mediated isothermal amplification (LAMP) uses 4-6 primers recognizing 6-8 distinct regions of target DNA. A strand-displacing DNA polymerase initiates synthesis and 2 of the primers form loop structures to facilitate subsequent rounds of amplification. LAMP is rapid, sensitive, and amplification is so extensive that reaction byproducts can be seen by eye, making LAMP well-suited for field diagnostics.
Produces long, branched DNA product
NEB Products
Bst 2.0 DNA Polymerase (NEB #M0537)
WarmStart® RTx Reverse Transcriptase (NEB #M0380)
WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA) (NEB #M1800)
WarmStart® LAMP Kit (DNA & RNA) (NEB #E1700)
NEB Products for LAMP
65°C
<250 nt
Visual
Lateral flow
Gel
Turbidity
WGA/MDA
Whole Genome Amplification & Multiple Displacement Amplification
Whole Genome Amplification (WGA) is a general term denoting methods that aim to amplify an entire genome. Multiple Displacement Amplification (MDA) is a WGA method that uses the strand-displacement activity of DNA polymerases such as phi29 or Bst.
phi29 DNA Polymerase (NEB #M0269)
NEB Products for WGA/MDA
30°C
N/A (WGA)
N/A
Pyrophosphatase, Inorganic (yeast) (NEB #M2403)
T7 Endonuclease I (NEB #M0302)
Deoxynucleotide (dNTP) Solution Mix (NEB #N0447)
SDA/NEAR
Strand Displacement Amplification & Nicking Enzyme Amplification Reaction
Strand displacement amplification (SDA) relies on a strand displacement DNA polymerase and a DNA nicking event. The nicking site is regenerated with each polymerase displacement step for repeated cycles of nicking and extension, the downstream strand is displaced available for amplification from the other end, resulting in exponential amplification. Nicking Enzyme Amplification Reaction (NEAR) is a similar amplification approach and powers point-of-care detection platforms.
Produces short, discrete DNA product
Bst 2.0 WarmStart® DNA Polymerase (NEB #M0538)
NEB Products for SDA/NEAR
60°C
Fluorescence
<100 nt
WarmStart RTx Reverse Transcriptase (NEB #M0380)
Nt.BstNBI (NEB #R0607)
RPA
Recombinase Polymerase Amplification
Recombinase polymerase amplification (RPA) and strand-invasion based amplification (SIBA) are isothermal amplification methods enabled through a recombinase enzyme that helps primers invade into double-stranded DNA.
Bsu DNA Polymerase, Large Fragment (NEB #M0330)
T4 Gene 32 Protein (NEB #M0300)
NEB Products for RPA
37°C
<1,000 nt
HDA
Helicase-dependent Amplification
Helicase-dependent amplification (HDA) employs the double-stranded DNA unwinding activity of a helicase to separate strands, enabling primer annealing and extension by a strand-displacing DNA polymerase. Like PCR, this system requires only two primers. HDA has been employed in several diagnostic devices and FDA-approved tests.
IsoAmp® II Universal tHDA Kit (NEB #H0110)
NEB Products for HDA
<150 nt
Tte UvrD Helicase (NEB #M1202)
Bst 2.0 WarmStart DNA Polymerase (NEB #M0538)
NASBA/TMA
Nucleic Acid Sequence-Based Amplification & Transcription-Mediated Amplification
Nucleic Acid Sequences Based Amplification (NASBA) and Transcription-Mediated Amplification (TMA) are both isothermal amplification methods that proceed through RNA. Primers are designed to target a region of interest; one of the primers must include the promoter sequence for T7 RNA polymerase at the 5´ end. NASBA and TMA reactions are utilized in a range of clinical diagnostics.
Produces short, discrete RNA & DNA products
Hi-T7® RNA Polymerase (NEB #M0658)
T7 RNA Polymerase (NEB #M0251)
Thermostable RNase H (NEB #M0523)
NEB Products for NASBA/TMA
40–55°C
Ribonucleotide Solution Mix (NEB #M0466)
